Molecular imaging

ABSTRACT

Disclosed are chemically-produced specific binding, &#34;molecular imaged&#34; sorbents which reversibly bind a preselected macromolecule by spacially matched multipoint interactions between functional groups synthesized on the surface of the sorbent and functional groups on the surface of the macromolecule. Also disclosed are methods of producing such sorbents. The sorbents typically are high surface area solids comprising surface binding regions which have charged groups, metal coordinating groups, hydrophobic moities, or various combination thereof anchored thereto and spaced in the mirror image of complementary interactive groups on a surface of the macromolecule.

BACKGROUND OF THE INVENTION

This invention relates to solid sorbents having surface defining sitescapable of selectively binding a preselected macromolecule, useful inthe separation of a target solute from a complex mixture and in varioustypes of analyses. The invention also relates to a family of synthetictechniques useful in fabricating such surfaces.

Adsorption of macromolecules such as proteins to surfaces involvesattraction at multiple sites through hydrophobic, electrostatic, andhydrogen bonding. Surfaces used in chromatographic packing materialstherefore have a high density of ionic, hydrophobic or hydroxylcontaining groups available for this adsorption process. The interfacebetween the surface and adsorbed proteins may cover between about 10-100surface groups on the sorbent, depending on the surface density of thecharged or other groups and on the size of the protein. Adsorptiontypically occurs through 5 to 10 groups on the surface of the protein,so there is a large excess of surface functional groups. As the surfacedensity of functional groups on a sorbent decreases, the strength ofprotein adsorption typically decreases rapidly. Although the number ofgroups on the sorbent surface is more than adequate for binding, thegroups are not distributed properly in space.

The effect is illustrated schematically in FIGS. 1A and 1B. In FIG. 1A,the accessible surface area of a protein, depicted at 10, has fivedispersed anion groups, all of which lie close to one or more cationgroups disposed at high density in a field on the surface 12 of theadsorbent. As shown in FIG. 1B, at lower surface density, the proteinwill be less avidly bound, as the spatial distribution of the anions onthe protein surface do not match up well with the positioning of thecations on the sorbent.

Of course, real behavior differs in several respects from theoversimplified situation depicted, as, for example, 1) charged groupsare randomly positioned on the sorbent, 2) adsorption occurs in threedimensions, e.g., the charge pair in the square shown in FIG. 1B may bespaced apart in a direction normal to the plane of the paper, 3) theprotein may have cation groups on its surface which will be repelled bythe cation surface and 4) there are other physical interactions at workin addition to electrostatic attraction.

This complimentary adsorption phenomenon is used most widely inchromatographic processes involving purification and analysis ofanalytes exploiting differential sorption properties of solutes in amixed solution. Those who manufacture chromatographic systems generallyseek to make the surface of the sorbent as homogeneous as possible, andto have a high density of functional groups. Complementarity is based onthe presence of a single set of functional groups on the sorbent surfacebeing complementary with a subset of the functional groups on theanalyte. In adsorption chromatography, for example, silanol groups atthe surface of silica are used to associate with solutes throughhydrogen bonding. This generally is achieved in an organic solvent wherehydrogen bonding is strong. In ion exchange chromatography, as notedabove, a charged surface interacts with a molecular species of oppositecharge through electrostatic interaction. The driving force forinteraction is based in part on enthalpic changes upon binding and inpart upon entropic effects from the displacement of water at the surfaceof both the sorbent and the sorbate. In reversed-phase and hydrophobicinteraction chromatography, the entropic effect is exploited to itsfullest as hydrophobic molecules are forced against the sorbent surfaceto minimize their hydrophobic contact area with the relatively polarsolvent. Immobilized metal affinity chromatography is yet anotherexample of the participation of complementary functional groups in theadsorption process. In this system, immobilized metal coordinationcompounds interact in the presence of metal such as zinc or copper withhistidine on an accessible exterior surface of a polypeptide. Thisassociation causes the differential adsorption of polypeptides based onnumber and spatial arrangement of histidines. All of these systemsexploit a surface having a random high ligand density. No attempt ismade to match specific structural features of the molecule withstructural features of the sorbent surface.

Affinity chromatography is based on exploitation of biological systemsto achieve intermolecular docking and adsorption. In this system, thesurface of the sorbent is caused to mimic a biological substance whichnaturally associates with a polypeptide. Affinity interactions generallyare based on multiple phenomenon including electrostatic attraction,hydrophobic interaction, hydrogen bonding, and stereochemical interfit.

Reversible binding interactions between pairs of biologicalmacromolecules such as ligands and receptors or antibodies and antigenshave been exploited widely to construct systems taking advantage of theexquisite specificity and affinity of these interactions. Affinitychromatography often involves the immobilization of specific bindingprotein, previously typically polyclonal antisera, but now commonlymonoclonal antibody, to a high surface area solid matrix such as aporous particulate material packed in a column. The feed mixture ispassed through the column where the target solute binds to theimmobilized binding protein. The column then is washed and the targetsubstance subsequently eluted to produce a fraction of higher purity.Solid material comprising such specific binding surfaces also are usedin immunoassay where immobilized binding protein is used to captureselectively and thereby separate an analyte in a sample.

There has been steady, sometimes dramatic improvement in methods forproducing specific binding protein useful in such contexts and forimmobilizing them on surfaces. Thus, monoclonal antibodies largelyreplaced polyclonal antisera obviating the need to purify the antibodiesfrom bleedings, enabling epitope-specific binding, and established atechnology capable theoretically of producing industrial quantities ofthese valuable compounds. More recently, advances in protein engineeringand recombinant expression have permitted the design and manufacture oftotally synthetic binding sites mimicking the antigen binding domains ofthe natural antibodies.

While this technology is very useful it is not without its drawbacks.The binding proteins are high molecular weight biological macromoleculeswhose function depend on maintenance of a tertiary structure easilyaltered upon exposure to relatively mild condition in use or storage.Furthermore, while it is now within the skill of the art to prepareantibodies or their biosynthetic analogs having specificity for apredetermined target molecule, the preparative technique aretime-consuming and costly, purification is difficult, and the techniquesfor immobilizing them onto surfaces at high density while maintainingactivity is imperfect. Furthermore, when such specific binding surfacesare used for the purification of substances intended for therapeutic orprophylactic use in vivo, they introduce a risk of contamination of theproduct by foreign biological material. This complicates qualitycontrol, increases the complexity of the design of a purificationsystem, and increases the expense and time required to obtain regulatoryapproval of the drug.

Molecular recognition is an important phenomenon in biological systems.The area involved in the interface between the surface and the analytecan be as small as 10 to 100 square Å in the case of amino acids andmonosaccharides and range to as large as thousands of Å in the interfacebetween polypeptides forming quaternary structure. At the level betweenabout 10-100 square Å surface area in the interface, man has beensuccessful in mimicking nature. This is the basis for modern affinitychromatography discussed above. However, the ability to discriminatecould be increased by using a broader surface area at the interface.

It is an object of this invention to provide rationally designed,stable, inexpensive to manufacture surfaces on solid materialscomprising a multiplicity of site which reversibly, noncovalently bindwith high specificity and affinity a preselected target molecule.Another object is to provide such materials adapted for use in varioustypes of analyses involving specific binding which heretofore have beenlimited to the use of immobilized macromolecules of biological origin.Still another object is to provide solids having surfaces containingspecific binding sites useful for both preparative and analyticalchromatographic separations, which, as compared with conventionalaffinity chromatography surfaces, are more durable, useful over agreater range of conditions, and less expensive to manufacture. Stillanother object is to provide a family of synthetic techniques whichpermit synthesis of rationally designed surfaces containing amultiplicity of regions which, through a combination of spatiallymatched electrostatic attraction, hydrophobic interaction, chelation,hydrogen bonding, and/or stereochemical interfit, are capable of bindingto any given macromolecular surface.

These and other objects and features of the invention will be apparentfrom the drawing, description, and claims which follow.

SUMMARY OF THE INVENTION

The invention relates to novel sorbents as compositions of matter andmethods of making a sorbent useful for binding a preselected molecule atits surface by complementary functional group interaction. Due to thiscomplementarity, there is a selective, reversible association betweenthe molecule and the surface. This association may be used in thepurification of the molecule, in its detection or quantitation, and inits removal from a complex system. The methods for making such specificbinding surfaces are termed herein "molecular imaging" methods. Thesurface is said to be an "imaged surface." Practice of the inventionprovides high surface area chromatography matrix material,molecular-specific sorbents, and catalytically active surfaces. Thesematerials are synthesized as disclosed herein by covalently adhering, ina way that is geometrically controlled at least in the directionparallel and preferably also in a direction normal to an underlyingsurface plane, a plurality of charged groups, hydrophobic groups, metalcoordination groups, and various combinations thereof, to form a mirrorimage of groups complementary to them on a molecular surface of a targetmacromolecule. These groups preferably are spaced about a hydrophilicundersurface rich in hydrogen containing groups and electronegativeatoms such as oxygen, nitrogen, phosphorus, or sulfur which take part information of hydrogen bonds.

More specifically, in a first aspect, the invention provides a solidmaterial defining a binding surface which comprises a multiplicity ofregions capable of selective binding of a preselected macromoleculehaving a plurality of ionizable groups spaced about its molecularsurface. Each of the regions comprise a plurality of charged moietiesbonded, preferably covalently bonded, to the surface or a coatingadhered to the surface, and disposed in spaced-apart relation within theregion in a mirror image and charged inverse of at least a subset of theionizable groups on the surface of the macromolecule. These regions bindthe preselected molecule preferentially to other molecules by virtue ofthe spatially matched electrostatic attraction between the surface ofthe molecule and the binding surface.

In preferred embodiments, the binding surface is substantially free ofbound charged moieties in excess of those which bind to the ionizablegroups on the preselected molecule. The binding surface preferablycomprises a coating adhered to the surface of a solid particulatematerial useful, for example, in chromatography, and comprising, forexample, particulate styrene divinylbenzene. The charged moieties maycomprise negatively charged groups such as carboxylate, sulfonate,phosphate, or phosphonate. Carboxyl groups currently are preferred. Thecharged moieties also may comprise positively charged groups such asprimary, secondary, tertiary or quaternary amines. These chargedmoieties preferably are bonded to the solid matrix or to an adherentcoating constituting the binding surface through flexible oligomericchains anchored to the underlying surface so that the spaced apartcharged moieties define a conformationally compliant charged surface,and the charged moieties are disposed at varying distances from thesurface of the underlying substrate so as to match, at least to someextent, surface topography of the preselected macromolecular species.Preferably, each binding region on the surface presents an interfacingsurface area of at least 50 square Å, preferably at least 500 square Å,and most preferably over 1000 square Å or more. An important advantageof the invention is that the interfacing area of binding can be muchlarger than that of an antigen-antibody interaction. The binding surfaceunderlying the spaced apart charged residues preferably is an oxygenrich hydrophilic polymer surface. Imaged surfaces may be synthesized toselectively adsorb various biological macromolecules and are well suitedfor selectively sorbing proteins such as natural or syntheticlymphokines, cytokines, hormones, growth factors, peptides, morphogens,enzymes, cofactors, ligands, receptors, antibodies and other valuableproteins and polypeptides. They may also be designed to sorb analogs ofintermediates in organic reactions thereby to produce catalytic surfacesmimicking the behavior of enzymes.

The spatially dispersed charged moieties bonded to the binding surfacemay be present in combination with one or more hydrophobic patchesdisposed at a location within the binding region which interface withone or more patches on the surface of the macromolecule. The surfacesalso may include one or more metal coordinating moiety disposed atlocations in each binding region to form, in the presence of acoordinating metal ion, metal coordinating bonds between thecoordinating moiety and an imidazole residue such as histidine exposedon the surface of a macromolecule.

In a second aspect, the invention provides a solid sorbent materialdefining a binding surface having regions which selectively bind apreselected organic macromolecule through one or more metal coordinatingbonds between the sorbent surface and imidazole residues spaced about amolecular surface of the macromolecule. Each region on the bindingsurface comprises one or more metal coordinating moieties, againdisposed in spaced-apart relation within the region in a mirror image ofat least a subset of the imidazole residues. In the presence ofcoordinating metal ions, the surface regions bind the preselectedmolecule preferentially to other molecules by multipoint spatiallymatched metal coordination bonds between the coordinating moieties onthe sorbent surface and imidazole residues, e.g., histidine residues, onthe surface of the preselected macromolecule. This type of surface canbind selectively with high affinity particularly well to proteins havingmultiple exposed histidine residues.

In still another aspect, the invention provides such a solid materialwhich defines a binding surface comprising regions which selectivelybind through multiple hydrophobic patches. Each region on the bindingsurface has plural hydrophobic moieties, surrounded by hydrophilicsurface, bonded to the binding surface and again disposed inspaced-apart relation within the region in a mirror image of at least asubset of the hydrophobic patches on the surface of the preselectedmolecule. Such imaged regions bind by spatially matched hydrophobicinteraction to the molecular surface of the preselected compoundpreferentially to others.

Imaged surfaces containing multiple regions which exploit variouscombinations of these effects, and especially those which extend oversurface area of 1000 square Å or more, provide powerful, stable bindingsystems approaching, equaling, or even exceeding the discriminatorycapabilities of the binding molecules of the immune system.

The preferred method of fabricating these molecular imaged surfaces alsocomprise an important aspect of the invention. Broadly, after selectingthe target macromolecule, the synthesis of the molecular image on thesurface of a solid is conducted by contacting a solution of thepreselected macromolecule with a specially derivatized activated surfaceproduced, for example, as disclosed herein, permitting or inducingreaction between certain groups on the surface of the preselectedmolecule and the derivatized surface, and then converting remainingreactive moieties on the derivatized surface to inactive form. Next, thecovalent bonds between the imaging molecule and the surface are cleaved,or the preselected imaging molecule is digested while leaving residuesof the macromolecule covalently bound to the surface. Then, the surfaceis "developed" to convert the remaining residues into matching,covalently attached, charged, hydrophobic, or metal coordinating groups,or by producing charge at each cleavage point. The optimal strategy forimaging a particular macromolecule may be discerned using computerizedprotein and other macromolecule modeling techniques as disclosed herein.

More specifically, appropriately spaced ionizable groups may be producedon a surface by providing as a starting material a solid having asurface layer of moieties covalently reactive with ionizable groups,contacting the surface layer with a preselected polyaminoacidmacromolecule under conditions in which the ionizable groups of themolecule react with the surface by multipoint formation of covalentbonds between at least some of the ionizable groups and the molecularsurface, and then digesting the amino acid polymer by hydrolyzingpeptide bonds, using strong base or enzymatic hydrolysis, leaving anamino acid residue, covalently bonded to the surface, at each positionwhere an ionizable group had reacted. Next, the amino groups or thecarboxylic acid groups of each of the bound amino acids is derivatizedto leave a charge opposite in sign and in space to the charge of theionizable groups on the surface of the preselected peptide bonded aminoacid polymer. This results in the production on the surface of spatiallydistributed charged groups in a mirror image and charge inverse of thereacted subset of the ionizable groups on the molecular surface.

The approach to producing spatially specific metal coordinatingcompounds is similar but distinct. In this case, a solid substratematerial having a surface layer of moieties covalently reactive with anorganic, nitrogen containing, polycarboxylic acid metal coordinatingcompound is required. This surface layer may be the same type of surfacelayer as is used in making electrostatic molecular imaged surfaces. Thismaterial is used in a heterogeneous reaction together with thepreselected molecule containing an imidazole residue and a metal ion,under conditions to produce multipoint formation of coordinated metalion links between at least some of the imidazole residues in thepreselect macromolecule and molecules of the coordination compound, andto produce covalent bonds between at least a subset of the covalentlyreactive moieties on the surface of the solid material and thecoordination compound. Next, the metal ions are removed from thereaction mixture, e.g., by chelation, to produce on the surface of thematerial a multiplicity of regions comprising plural, covalently bondedmetal coordinating compound molecules spaced apart within the regions inthe mirror image of the imidazole residues on the macromolecule.

The approach to producing hydrophobic surface in the binding regions,appropriately located to interact with the macromolecule by hydrophobicattraction, involves prereacting the preselected molecule with anamphipathic molecule. The term amphipathic molecule, as used herein,refers to a molecule comprising a hydrophobic moiety, such as ahydrocarbon, halocarbon, or aromatic residue, which associates with ahydrophobic patch on the target molecule, and a covalently reactivegroup, such as an amine, carboxylic, aldehyde, or epoxy residue, adaptedto react with the activated surface. One then provides a startingmaterial having, e.g., the same covalently reactive hydrophilic surfaceas is used to derivatize with charged or metal coordinating groups, andreacts this solid starting material with the amphipathic moleculepreselected molecule complex, held together by hydrophobic-hydrophobicinteraction. This reaction produces a sorbent comprising hydrophobicbonding interaction sites, which associate with at least a subset of thehydrophobic patches on the surface of the preselected macromolecule, andwhich are covalently bonded to the surface through the covalentlyreactive moieties on the solid binding surface and the covalentlyreactive groups of the amphipathic molecules. The preselected moleculethen is desorbed from the surface by breaking thehydrophobic-hydrophobic attraction to yield a surface comprising regionswherein plural, covalently anchored hydrophobic moieties are spacedwithin a hydrophilic field in the mirror image of the hydrophobicpatches on the molecular surface of the preselected macromolecule.

In preferred aspects of the method, high surface area solid material isused, e.g., a perfusive matrix material such as is disclosed in U.S.Pat. No. 5,019,270, and the first step in the manufacturing process isto produce a uniform, adherent, hydrophilic, derivatizable coating aboutthe entirety of the surface of the solid matrix, e.g., in accordancewith the method disclosed in U.S. Pat. No. 5,030,352. Next, the coatingis derivatized with oligomer chains of reactive monomers, e.g.,comprising aldehyde or epoxy groups to produce a field of activefilaments. A solution of the imaging macromolecule next is placed incontact with the derivatized, reactive surface of the matrix. As themolecule comes in contact with the derivatized surface, nucleophilicamine groups exposed on the molecule surface covalently react with asubset of the epoxy or aldehyde groups on the surface of the matrix. Aschiff base is formed in the case of aldehyde coupling which is reducedto a secondary amine. The support-protein complex next is hydrolyzed,breaking peptide bonds linking amino acids in the protein, and allremaining epoxy groups in the case of base catalyzed hydrolysis of theepoxy support matrix. This leaves a single amino acid covalently bondedto the surface through its amino side chain leaving a free amino groupand a free carboxylic acid group at each point where amines on thepolypeptide reacted with the activated surface. To make an imaged anionsurface, one derivatizes the amino terminal of the bound amino acids,e.g., by converting them to amidates using an anhydride, thereby leavingcarboxylic acid groups and their characteristic negative charge at eachpoint on the surface corresponding to amino groups on the surface of thepolypeptide.

Sorbents comprising cations covalently bonded and spaced in the mirrorimage of plural exposed anions on the molecular surface of amacromolecule can be produced with an analogous strategy using differentchemistry. In this case, one starts with a sorbent surface derivatizedwith, for example, a terminal amine group having an adjacent vicinalhydroxyl group (--CHOH--CH₂ -NH₂). Upon exposure of the imaging moleculeto the surface of in the presence of a water soluble carbodiimide suchas 1-ethyl-3(3-dimethylaminopropyl)-carbodiimide, amide bonds are formedbetween carboxylate ions on the surface of the macromolecule and theamine groups on the surface of the sorbent. Oxidation with periodatecleaves the remaining --CHOH--CH₂ -NH₂ to produce a bonded aldehyde(--CHO) group. This group will react with any lysine, arginine, orN--terminal amino groups on the macromolecule that are located at thesorbent-macromolecule interface, forming a schiff base. Sodiumborohydride is then used to convert the residual surface aldehyde groupsto primary alcohols and schiff bases to secondary amines. Nexthydrolysis in, for example, potassium hydroxide, leaves an imagedsorbent surface having cationic amine groups bonded to the sorbentsurface in locations opposite the anionic carboxylic acid groups on theimaged macromolecule. Electrostatic imaging of anionic species iscarried out with molecules that have an excess of anionic functionalgroups. For this reason, only a smaller number of cationic amino acidsare bonded to the surface in this process and will have little effect onthe adsorption of anionic species.

To produce mirror imaging points of hydrophobicity to inducehydrophobic-hydrophobic interaction, i.e., a specific binding reversephase sorbent, one mixes together the imaging molecule having one ormore hydrophobic patches on an exterior surface and an amphipathicmolecule comprising a hydrophobic moiety, e.g., a hydrocarbon orhalocarbon, and an opposing moiety comprising a group covalentlyreactive with an activated surface of the type described above. Forexample, the covalently reactive group may be an amine or carboxylategroup. When the amphipathic molecule and imaging molecule are broughttogether in relatively hydrophilic media, the hydrophobic regionsassociate to exclude water molecules between their hydrophobic contactsurfaces. This complex is then reacted as disclosed above such that, atthe end of the synthetic scheme, the hydrophobic end of each amphipathicmolecule extends through a covalent linkage from the surface of thesorbent and is located in space such that it interfits with ahydrophobic patch on the surface of the imaging molecule. This techniqueis particularly powerful when the molecule is electrostaticallyattracted by charge on the surface.

The preferred approach to produce sorbent material having an imagesurface which binds selectively to macromolecules having imidazoleresidues on its surface, e.g., proteins having exposed histidineresidues, involves reacting the imidazole containing macromolecule inthe presence of copper or other metal ion and a metal coordinatingcompound such as iminodiacetic acid (IDA). This results in formation ofa copper coordination complex between imidazole moieties on the surfaceof the target protein and the IDA moieties. The nucleophilic nitrogen inthe iminodiacetic acid moiety then can be reacted with aldehyde or epoxygroups in the reaction schemes noted above so that, at the conclusion ofthe synthesis, an IDA moiety is covalently bonded to the surface of thesorbent at the precise location in space matching the imidazole residueson the imaging macromolecule. This technique also most preferably isused with charge group matching, but may be used separately.

It will be seen that a key to synthesis of molecular imaged surface isto orient an appropriate surface of the target molecule in face-to-facerelation with the sorbent surface. In accordance with the invention, therelationship of the imaging molecule to the sorbent surface may bepermitted to occur relatively randomly, in which case a "polyclonal"sorbent will be produced, i.e., one in which the multiplicity of bindingregions on the sorbent surface contain the mirror images of differentpresented surfaces of the macromolecule. However, sorbents having ahigher frequency of regions imaged to a given face of the imagingmolecule can be produced by using several strategies, e.g., taking stepsto assure more consistent orientation of the molecule during earlystages of the imaging process, or using peptide analogs of a surfaceregion of the macromolecule sought to be imaged. Thus, for example, thepresence of anti-chaotropic salts such as sodium sulfate in solutionwith the macromolecule at the imaging stage will induce the morehydrophobic face of the target macromolecule to contact the activatedsorbent surface. Alternatively, one may include anionic or cationicgroups on the activated surface to "dock" a surface of the macromoleculerich in moieties of the opposite charge by electrostatic attraction.

BRIEF DESCRIPTION OF THE DRAWING

FIGS. 1A and 1B are drawings schematically illustrating the nature ofadsorption of macromolecules onto a high density and low densitycationic surface, (anion exchange) respectively.

FIGS. 2A, 2B, and 2C are illustrations which depict the relationship ofa protein or other large macromolecule and a surface imaged as disclosedherein. FIG. 2A and 2B are "plan view" illustrations looking through anadsorbed protein onto the molecular imaged surface. FIG. 2C is anillustration taken in cross-section showing the nature of the molecularimaged sorbent and the protein adsorbed thereon, and illustrating howthe use of oligomeric filaments extending from the sorbent surface canaccommodate varying molecular topology on the surface of proteins.

FIGS. 3A, 3B, and 3C depict exemplary activated surfaces of the typeuseful as a starting point in the synthesis of molecular imaged surfacesof the invention.

FIGS. 4A-4E are molecular diagrams useful in explaining how to make amolecular imaged surface having anionic groups spaced thereabout in themirror image of cationic groups on a preselected protein, starting withthe activated surface illustrated in FIG. 3A.

FIGS. 5A-5D are molecular diagrams similar to those in FIG. 4 but usingthe activated surface of FIG. 3B in place of 3A, and ending with animaged surface comprising appropriately spaced (both horizontally andvertically with respect to the substrate) anionic charges in themolecular image of the preselected molecule.

FIGS. 6A-6E are molecular diagrams illustrating how to make a molecularimaged surface beginning with an activated substrate comprising a highdensity of anionic, carboxylic acid groups whereby the imaging moleculeis oriented with respect to the surface by electrostatic forces, i.e.,presents its most positively charged surface to the sorbent.

FIGS. 7A-7E are molecular diagrams illustrating how to make a molecularimaged sorbent having plural, spaced-apart cationic groups, andbeginning with an imaging macromclecule having plural anionic groupswhich are attracted electrostatically to the surface in the first stageof the synthesis.

FIG. 8A illustrates a macromolecule having a hydrophobic patch, e.g., aprotein having a region high in amino acids with aliphatic or aromaticside chains, in association with two amphipathic molecules comprising ahydrophobic region linked to a primary amine, and disposed in contactwith an activated surface of the type illustrated in FIGS. 3B and 5A.FIG. 8B shows the imaged surface resulting from the starting point of 8Ahaving both anionic groups and a hydrophobic patch disposed in themirror image of the protein depicted in FIG. 8A.

FIG. 9A depicts a macromolecule having a pair of amine side groups andan imidazole side group complexed through a metal ion to animinodiacetic acid moiety, with the macromolecule and complex disposedadjacent an activated surface of the type illustrated in FIG. 3A. FIG.9B illustrates a molecular imaged sorbent made by following a preferredsynthesis disclosed herein from the starting point of FIG. 9A, and FIG.9C shows the imaged surface of FIG. 9A comprising a pair of anionicgroups disposed in the mirror image of the amine groups on the surfaceof the macromolecule and an iminodiacetic acid moiety properly disposedwith respect to the surface of the imaging protein to form a metalcoordinating bond with its imidazole side group and its multipointelectrostatic attraction.

FIGS. 10A-10E are elution profiles illustrating the properties of animaged column. In each case, the shaded peak represents the imagingmolecule, here Lysozyme.

FIG. 11 depicts superposed elution profiles (pH=6.2, 0.0-1.0M NaClgradient, 5 ml/min in 5 min.) of the same Lysozyme/Cytochrome C mixtureson a Lysozyme imaged column and on a strong cation exchange column.

FIG. 12 is a flow chart useful in describing how to optimize imagedsurfaces using computer aided design principles.

Like reference characters in the respective drawn figures indicatecorresponding parts.

THE NATURE OF THE MOLECULAR IMAGED SURFACE

The highest specificity of binding between a biomolecule and a surfacecurrently is achieved using affinity interaction between, for example,antibodies and antigen, receptors and ligands, lectins and theirreceptors, avidin and biotin, etc. Both strength and specificity areimportant in such specific binding reactions. Affinity based systemsoften involve binding constants in the range of 10⁶ to 10⁹ M⁻¹, and canbe as high as 10¹⁵ M⁻¹. Surfaces capable of specific, tight-binding witha preselected molecule currently are produced by exploiting naturallyoccurring biological binding systems. These systems in turn exploit acombination of electrostatic interaction, hydrophobic-hydrophobicinteraction, hydrogen bonding, and stereospecific interfit to achievehigh affinity selective binding.

This application discloses how specific binding sites can be produced onsurfaces without resort to the production, collection, and attachment ofbiological binding molecules such as antibodies or receptors. Binding tosorbent surfaces of the invention is selective, i.e., shows a preferencefor the imaged molecule versus other molecules, and reversible, i.e.,involves no covalent bonding. Selective binding, as used herein, meansthat the surface binds the imaging macromolecule in preference toothers. Reversible binding, as used herein, means that binding isachieved without formation of covalent bonds. The chemically definedbinding sites of the invention tend to be more stable, resist leachingfrom the surface, can be reproducibly synthesized, need not exposeproduct to biological materials, and obviate the risk of contaminationof product by biomolecules incident to product purification. Thisprocess, a type of rational surface design, involves the creation of aspecific binding sorbent surface, herein called an "imaged" surface,which is complementary to a surface of a molecule of interest,hereinafter referred to as the "imaging" molecule.

Adsorption of molecules at a surface is based on the existence ofcomplementarity between at least some functional groups on the moleculeand those at the surface. FIGS. 1A and 1B, discussed above, exemplifyadsorption of a protein onto the surface 12 of a strong cation resin(FIG. 1A) and a weak cation resin (FIG. 1B). As should be apparent, thehigh density cation sorbent of FIG. 1A will result in a more tightlyadhered protein as there is a high frequency of multipoint electrostaticattraction between negative groups on the surface of the protein andpositive groups on the sorbent surface 12. Neither the weak nor strongcation exchange sorbent exhibits specificity for any givenmacromolecule.

In contrast, FIG. 2A depicts a region of a sorbent surface 12 containingonly five cationic groups. However, as illustrated, these groups arearranged on sorbent surface 12 such that they are opposite in space tothe five negative charges on the surface of the protein 10. Thisdistribution of positive charges in this region of the surface, becauseit represents the mirror image of the negative charges on the surface ofthe protein 10, specifically bind protein 10 in preference to otherproteins where the charge distribution does not match. Thus, althoughthe charge density of the cationic moieties in the sorbent surface 12 ofFIG. 2A is less than the charge density in FIGS. 1A or 1B, protein 10will adhere to the imaged surface of FIG. 2A with greater affinity, andfar greater specificity, than it will to the surfaces in FIG. 1.

FIG. 2B illustrates still another principle of the molecular imagingtechnology disclosed herein. Specifically, in FIG. 2B, a differentprotein 14 is depicted as having a pair of hydrophobic patches 16, 16',three negatively charged groups, and a surface histidine residue. Thehistidine residue has an imidazole side chain which, as is known, can beattached to a metal coordinating compound by complexation with metalsuch as copper or zinc. In FIG. 2B, the molecular imaged surface 12comprises a corresponding pair of hydrophobic patches 17, 17-, threeappropriately spaced positive ions, and a covalently linkediminodiacetic acid (IDA) metal coordinating molecule disposed oppositethe position of the histidine residue on the protein 14. As illustrated,the protein 14 will associate with imaged surface 12 of FIG. 2B withhigh specificity and affinity. When the protein attains its properorientation, three positive charges properly spaced on the surface ofthe sorbent 12, a pair of hydrophobic patches, and, in the presence ofcopper ions, a metal coordinating bond, all acts simultaneously to holdprotein 14 in position. It will immediately be apparent that, forexample, surface 12 of FIG. 2B will readily discriminate betweenmacromolecule 14 and macromolecule 10.

FIG. 2C illustrates still another aspect of the molecular imagingtechnology disclosed herein. This drawing schematically illustrates a"cross-sectional"0 view through an imaged surface 12 and yet anotherdifferent protein, here depicted as 18. The bottom surface 20 of theprotein 18 comprises peaks and valleys, or a molecular topology, definedby the three dimensional structure of the macromolecule. Viewed fromleft to right, the surface of the protein comprises first a pair ofcationic groups, e.g., amine side chains in a protein such as those onthe lysine or arginine amino acid residues, a histidine residue disposedin a "valley" on the protein surface, a hydrophobic patch 16, and ananionic group, e.g., a carboxylic acid side chain such as is present onamino acid residues like aspartic acid or glutamic acid. In the sorbentof FIG. 2C, random length oligomer chain "filaments" covalently bondeddirectly to the matrix or to an adherent coating on the matrixcomprising the sorbent surface 12, extend upwardly and have appendedcharged groups opposite in sign to those on the face 20 of the protein18, a metal chelating group disposed opposite the histidine residue, anda hydrophobic moiety 17 disposed opposite the hydrophobic patch 16 onprotein 18.

From the foregoing it should be apparent that proper disposition ofcharges, hydrophobic patches, and metal coordination groups, both withinthe plane of a sorbent surface 12 and in a direction more or lessperpendicular to the plane, if embodied in a real structure, couldproduce chemical, as opposed to biological, binding sites of highspecificity and affinity. In this case the imaged surface is a copy,counterpart, or likeness of the target molecule displaying matchingopposite charge, matching hydrophobic patches, and/or matching metalchelating points which together interact chemospecifically with theimaged molecule and bind selectively and reversibly to the molecule, orat least display significant preferential adsorption of the imagedmolecule from a complex mixture.

Several approaches have been envisioned to achieve these goals. Thepresently preferred approach involves reacting the target macromoleculewith an activated surface and leaving behind complementary functionalgroups. The remainder of the specification will disclose how to make anduse such molecular imaged surfaces, and will discuss certain propertiesof such materials.

THE NATURE OF THE SOLID MATRIX

Sorbents having molecular imaged surfaces produced in accordance withthe invention have many uses. Chief among these is affinitychromatography purification procedures, activated sorbents for theremoval of a target molecule from a mixture, e.g., a toxin from food,and specific binding assays such as are used widely to detect thepresence or concentration of biological molecules, toxins, contaminants,drugs and the like in samples such as water, body fluids, and variousplant and animal matter extracts. In many of these uses the solidsubstrate, or matrix, ideally should have as high a ratio of surfacearea to volume as is practical. Since it often will be desirable totransport aqueous solutions containing biological molecules such asproteins, carbohydrates, lipids, steroids and the like into contact withthe surface to selectively bind or to induce a chemical change in acomponent in the liquid phase, it is often advantageous to use a rigidsolid having a uniform hydrophilic surface and a geometry which permitsconvective transport of solutes to the imaged surface. A rigid, highmechanical strength material permits high pressure flow withoutcrushing. Perfusive matrices are preferred. Methods for making perfusivematrix materials, the nature and unique geometry of these materials, andvarious of their advantages are disclosed in detail in U.S. Pat. No.5,019,270 issued May 28, 1991 and assigned to the owner of thisapplication. The preferred material for fabricating perfusive matricesis polymeric material such as polystyrene divinylbenzene, preferablysynthesized as disclosed in the above-referenced U.S. Patent inparticulate form. There are various ways of providing on the surface ofthe inert and hydrophobic styrene based matrix material a hydrophiliccoating well suited for interaction with aqueous solutions of biologicalmacromolecules. The currently preferred methods for providing suchcoatings are disclosed in U.S. Pat. No. 5,030,352 issued Jul. 9, 1991,and assigned to the Purdue Research Foundation of West Lafayette, Ind.The '352 patent discloses how to provide an adherent, crosslinkedhydrophilic, easily derivatized coating onto the surface of particulateand other types of matrix material. The coatings are compatible withprotein solutions and are extraordinarily versatile, permitting varioustypes of activated groups, oligomers, polymer chains and the like to befixed to the surface as desired. The disclosures of both the foregoingpatents are incorporated herein by reference.

Three exemplary activated surfaces suitable as starting points for theproduction of the imaged surfaces of the invention are disclosed,respectively, in FIGS. 3A, 3B, and 3C. The first of these represents aportion of a solid matrix 12, shown in cross-section, having a highdensity of epoxy groups covalently bound to a hydrophilic surfacecoating adhered to matrix material 12, for example, in accordance withthe procedure disclosed in the above-referenced Purdue patent. Matrixmaterial of this type may be produced from POROS® brand chromatographymatrix material, e.g., POROS® OH, and are available commercially asPOROS® EP from PerSeptive Biosystems, Inc., of Cambridge, Mass. Epoxygroups react with amine groups to produce an alcohol group and asecondary amine covalent linkage. The alcohol group contributes to thehydrophilicity of the surface. The secondary amine linkage forms astrong covalent bond which is exploited as disclosed below to makevarious types of molecular imaged surfaces.

FIG. 3B discloses another type of activated surface, preferred in manyinstances, comprising an oligomer of acrolein (acryloylaldehyde), whichis characterized by a hydrocarbon backbone having aldehyde groups (CHO)branching from alternate carbon atoms. This type of activated surface,having oligomers ranging from 1 to 20 monomer units, i.e., a, b, c, d,and e are between about 1 and about 20, can be produced from POROS®-OH,commercially available from PerSeptive Biosystems, Inc., as disclosedherein, by reaction with acrolein in the presence of cerium. Aldehydegroups also react readily with primary nitrogen atoms to producesecondary nitrogen linkages and water in the presence of sodium cyanogenborohydride. Other types of aldehyde activated surfaces are availablecommercially, e.g., POROS® AL depicted in FIG. 3C. Both the epoxy groupsand the aldehyde groups can be further derivatized to form, for example,hydroxyls, carboxylic acid groups, or amine groups using conventionalchemistry. These may be used as starting materials in various syntheticschemes as disclosed herein to produce molecular imaged surfaces.

An important aspect of the activated surface 12 of FIG. 3B is that thesurface presents a very high density of active aldehyde groups presentnot only over the entirety of the surface of substrate 12 but alsoextending away from the surface. Each filament comprises a series ofaldehyde side groups appended from a flexible hydrocarbon chain.Reactive moieties on the surface of the imaging molecule can react withthe aldehydes buried within or adjacent the surface of the field offilaments, as dictated by the surface shape of the imaging molecule.Furthermore, the filaments can flex and bend laterally to conform to ashape as required by making minor spatial adjustments. This type ofactivated surface, i.e., a surface having chemically active groupsdisposed on oligomer units extending upwardly from the surface, permitssynthesis of molecular imaged surfaces which can approximate or matchthe peaks and valleys on the surface of the imaging macromolecule asillustrated in FIG. 2C. It also assures multipoint formation of chargedor other groups.

It should be noted that the epoxy and aldehyde groups shown in FIG. 3are illustrative and preferred, but are by no means the only such groupsthat can be used. As will be apparent from the disclosure below, thenature of the activated surface groups can vary widely, depending on theparticular imaging chemistry used in the manufacture of the imagedsurface.

One important chemical feature of these starting materials is thesurface density of the surface anchored active groups. If, for example,a pair of charges disposed on a surface of a macromolecule to be imagedare five angstroms apart, then the active groups on the sorbent surfacemust be at least this close together to be useful in a molecular imagingprocess. On the other hand, a starting material having 9 or 10 activegroups per 100 square angstroms would be operative, although perhaps notoptimal, in imaging a molecular surface of, for example, 2000 squareangstroms, involving spaced apart charge or other surface features atleast 10-20 Å apart. It thus can be seen that the surface spacing ofactive groups on an imageable surface is directly analogous to grainsize in photographic surfaces, and that different surface densities mayhe used, depending on the resolution required.

THE IMAGING MOLECULE

Essentially any macromolecule may be imaged in accordance with theprocedures disclosed herein. The term "macromolecule," as used herein,refers to molecules having an imageable surface area of at least 50square Å. Proteins are currently preferred. Smaller peptides may also beused, and certain of the procedures disclosed herein may be used to formmolecular images of glycoproteins, polysaccharides, polynucleic acidsand other large molecules. Generally, the interfacing area of the imagedsurface and the imaging molecule (i.e., the area of interface betweensorbent and sorbate) should be at least about 50 square Å, morepreferably 100 Å, and often will exceed 1,000 Å.

It generally is preferable to limit the number of distinct surfaces on agiven macromolecule imaged in a given synthesis. This is because itwould be possible to create 10-20 different images of the surface of amacromolecule and that each could have a different binding constant. Itis also important, particularly in the case of proteins, to avoid duringthe imaging stage high concentrations of organic solvent, extremes inpH, or elevated temperature. All of these tend to alter the threedimensional structure of the protein or other imaging biologicalmolecule and to create a false image of the molecule, not reflective ofits native character.

An important aspect of molecular imaging therefore involves theorientation of the imaging molecule with respect to the surface to beimaged. When using the covalent immobilization synthetic route disclosedherein, molecular orientation can be achieved by using ananti-chaotropic salt such as sodium sulfate, to drive the protein to thesurface and promote hydrophobic interaction. Alternatively, chargegroups can be included at the surface to orient the imaging molecule ina naturally most favored binding conformation, i.e., one presenting amolecular face rich in the opposite charge.

Another approach to promoting homogeneous imaged binding regions on thesorbent surface is to use peptide analogs of a surface region of thetarget protein as the imaging molecule. Thus, digested samples of theimaging protein, or randomly generated peptides, may be screened, forexample, by affinity chromatography using monoclonal antibody, or usingan imaged surface produced as disclosed herein, to obtain a short, e.g.,5-20 amino acid, peptide which mimics the charge or other surfacefeature distribution of the imaging protein. Methods for producing suchpeptides are known in the art. Alternatively, rapidly growing data basesstoring X-ray diffraction and NMR data from various macromolecules ofimportance, and programs which display images of proteins and the likebased on such data and on amino acid sequence information, may be usedto determine sequence of a peptide which mimics the surface structure ofa given macromolecule. Use of such peptides as the imaging molecule maybe preferable for cost purposes when synthesizing large quantities ofimaged sorbent. They also can provide a source of analogs of short-livedintermediates useful in the preparation of catalytic surfaces, and inany event provide a means of promoting image homogeneity by verysignificantly reducing the number of surfaces available for imagingduring manufacture of the sorbents of this invention.

PREPARATION OF A MOLECULAR IMAGED ANIONIC SURFACE

In one embodiment, a molecular imaged surface is produced by contactinga protein comprising plural exposed lysine or arginine residues, withtheir characteristic primary amine side chains, with an epoxide surfacesuch as is illustrated in FIG. 3A, or with the aldehydegroup-derivatized surface of FIGS. 3B or 3C. After reaction between thealdehydes or epoxides and the amine groups, the protein is digested withstrong base, or enzymatically using a mixture of proteolytic enzymes,pronase, or the like, to leave only the corresponding lysine or arginineamino acids at the precise relative locations of these residues in theprotein. The positively charged amines are neutralized through acylationleaving a negatively charged carboxyl group at the exact location of apositively charged amine on the imaging protein. Surface hydroxylgroups, which may be esterified during the acylation step, may beconverted back to hydroxyl form by hydrolysis. Such an imaged surfacewill bind the imaging molecule selectively and reversibly throughmultipoint electrostatic attraction while all other proteins willinteract only very weakly as if they were encountering a very weak anionsurface.

Details of how the foregoing synthetic technique is conducted are shownin FIGS. 4A through 4E. Referring to the drawing, FIG. 4A illustrates anactivated surface 12 derivatized with plural epoxy groups and, disposedin solution and oriented close to surface 12, a protein, here depictedas amide-bonded amino acids including, for purposes of illustrating thetechnology of the invention, a central arginine residue flanked byintervening amino acid sequences and a pair of lysine residues. Asshown, the lysine residues comprise a side group consisting of C₄ H₈--NH₂ ; the arginine residue also has a side chain terminating in an NH₂group. The purpose of the synthetic procedure is to provide on thesurface 12 negatively charged moieties located in space about surface 12such that they match the location of the NH₂ groups pendant from theside chains of the lysine and arginine residues constituting a portionof the surface of the imaging protein. For ease of explanation, in FIG.4B and following, the protein backbone is represented simply by a lineextending horizontally, and only the side groups are identified.

AS shown in FIG. 4B, amine groups react with adjacent epoxy groupsforming covalent bonds through secondary amines linking surface 12 andthe protein. In the presence of weak base, such as sodium phosphate,pH:9, unreacted epoxy groups are opened to form hydrophilic dihydroxylcompounds. Next, the reaction mixture is treated with strong base suchas KOH so as to thoroughly hydrolyze the protein. One to three normalpotassium hydroxide is suitable for this step. Proteolytic enzymes mayalso be used. The result is shown is FIG. 4C, wherein only the twolysine and single arginine residues remain. Note the molecular structureon the termini of the covalently-linked chains extending from surface 12comprise the amino and carboxyl groups characteristic of amino acids.Next, the intermediate imaged surface illustrated in FIG. 4C is treatedwith, for example, acetic anhydride (CH₃ CO)₂ O in at appropriatesolvent such as pyridine, to acylate the amine groups. This results inderivatization and removal of the positive charge region of the aminoacid as shown in FIG. 4D, leaving behind the negatively-chargedcarboxylic acid groups located precisely opposite the amine groups onthe protein originally used to initiate the imaging procedure.

FIG. 4E illustrates the function of the imaged surface. As shown, theimaging protein presented together with other solutes in solution, whenencountering the imaged surface, binds preferentially as the amine groupin the lysine and arginine side chains "dock" with the carboxylic acidgroups by electrostatic attraction. Thus, the relationship of themolecular imaged surface and the imaging protein is as illustrated inFIG. 2A, i.e., selective sorption occurs by virtue of spatially-matchedanionic and cationic groups attached respectively to the imaged surface12 and the surface of the imaging molecule.

Note also in FIG. 4E that the surface 12 is covered with plural OHgroups. These can take part in hydrogen bonding and can increase theaffinity constant of binding between the imaging macromolecule and theimaged surface.

Referring to FIG. 5A through 5D, another series of molecular diagramssimilar to those set forth in FIG. 4 are shown which differ from FIG. 4in that substrate 12 is an aldehyde derivatized starting material of thetype illustrated in FIG. 3B. As shown in FIG. 5A, as the amine group of,for example, a lysine side chain, comes into contact with an aldehydegroup pendent from a filament some distance from the substrate 12, itreacts to form a secondary amine linking the protein to the surface asshown in FIG. 5B. Residual aldehyde groups are reduced to primaryalcohols by sodium borohydride. In the presence of strong base, thepeptide bonds linking the amino acids of the protein together arehydrolyzed. This results in a structure such as illustrated in FIG. 5Cin which, at each location where the protein had an amino side chain, anamino acid residue remains with its characteristic amine and carboxylicacid groups. The structure next is treated to acylate the amine groupsusing an acylating reagent such as acetic anhydride to produce thestructure of FIG. 5D having a negatively charged carboxylic acid locatedon surface 12 in position to mate with the various amine groups on theexposed side chains of the imaging protein.

As noted above, orientation of an imaging molecule prior to or duringthe reactive imaging step can be accomplished by either hydrophobic orelectrostatic interaction. An advantage of the electrostatic interactionis that it will provide an orientation which will maximize the number ofinterfacing charged sites involved in the synthesis. FIG. 6 illustratesone example of this type of reaction scheme which, in this case, allowselectrostatically oriented reactive imaging in production of the imagedsurface comprising plural, spaced apart anions.

FIG. 6A depicts a protein, here shown simply as lines terminating inamino side groups, interfacing with a reactive surface 12, herecomprising alphacarboxyl beta hydroxylic filaments extending upwardlyfrom surface 12. The negative charged polarity of the carboxyl groupsand the positive charged polarity of the amine groups on the proteininteract to settle the molecular surface of the protein into theactivated substrate surface during the imaging procedure. Unlike theprevious examples where essentially instantaneous reaction occurs uponcontact between an amine group and an epoxy or aldehyde group, in thisinstance no reaction occurs spontaneously, and equilibrium can beestablished between the imaging molecule and the surface to be imaged.This promotes production of relatively few separate molecular surfaceimages, i.e., tends to make all of the binding regions more nearly alikein their distribution of charge, and tends to orient the imagingmolecule with its most positively charged surface interfacing thesorbent.

In the presence of EDAC, peptide bonds are formed between the aminegroups and the carboxylic acid groups on the sorbent surface as shown inFIG. 6B. Next, in the presence of periodate, free carboxylic acid groupsare converted to aldehyde groups (FIG. 6C) and then in the presence ofsodium borohydride to alcohol groups (FIG. 6D). Next, strong base suchas KOH is used to hydrolyze all peptide bonds leaving carboxylic acidgroups covalently bonded to the now imaged surface, spaced thereabout inthe mirror image of the amine side groups on the imaging protein.

PREPARATION OF A MOLECULAR IMAGED CATIONIC SURFACE

FIGS. 7A through 7E illustrates how to make still another embodiment ofthe imaged surface of the invention. In this case, again, the imagingmolecule is steered to the surface by electrostatic forces such that theimaged surface of the protein is the area which is most anionic.Formation of the surface begins when a protein, here shown ascomprising, from left to right in FIG. 7A, an amino acid such asaspartic acid having an anionic carboxylic acid side chain, and an aminoacid such as glutamic acid with another carboxylic acid side chain, anda lysine having a cationic amine group on its side chain. Again, theimaging protein is permitted to reach equilibrium with the surface suchthat carboxylate groups on its side chains are electrostaticallyattracted by primary amine groups covalently attached directly to thematrix 12 or to a coating adhering to the matrix. An activated surfacecomprising a field of amine groups can be synthesized using a variety oftechniques. Alternatively, conventional, commercially available,polyimine or polyamine cation exchange resins may be used.

Negative charge on the surface of the protein is attracted to positivecharge on the surface of the activated surface. As indicated by adouble-headed arrow on the right of FIG. 7A, interfacing amine groupswould repel one another. Treatment of the reacting system with EDACproduces amide bonds between carboxylic acid side groups and the aminegroups on the sorbent as shown in FIG. 7B. Strong oxidation in periodate(IO₄) liberates methylamine from the surface and coverts the terminalalcohols into aldehyde groups as shown in FIG. 7C. Schiff base formationcan occur between the resulting aldehyde group and the primary aminegroup on the lysine side chain provided the density of the aldehydegroups is high enough so that an amine group is in close proximity. Upontreatment with sodium borohydride, the aldehyde groups are converted toalcohol groups, and the schiff base is converted to secondary amine asshown in FIG. 7D. Next, strong based hydrolyzes all peptide bondsleaving, as shown in FIG. 7E, amine group covalently linked to thesurface 12 and disposed opposite the carboxylate groups of the sidechains of the imaging protein. Where imaging protein originally had anarginine or lysine residue, it becomes linked with the imaged surface.

FIGS. 8A and 8B illustrate the method of synthesis of an imaged sorbenthaving anions and hydrophobic groups covalently linked to the sorbentsurface and disposed so as to cooperatively attract a protein having onits molecular surface a plurality of cations and a hydrophobic patch.FIG. 8A shows an activated surface 12 of the type illustrated in FIG. 3Bcomprising filaments extending from the surface, each of which haveplural pendent aldehyde groups. The imaging protein here is depicted ashaving a hydrophobic patch 16 disposed between a lysine and an arginineresidue.

Prior to mixing the aldehyde activated surface and the protein togetherto begin the imaging step, amphipathic molecules, here illustrated asamine soaps having a hydrophobic e.g., hydrocarbon, tail 17 and acovalently linked amine group, are mixed together under conditions inwhich the hydrophobic portion of the soap molecules associate and becomeembedded in the hydrophobic patch 16 on the surface of the protein.Thereafter, imaging and subsequent synthesis of the image surface isconducted essentially as disclosed above with respect to FIGS. 5Athrough 5D. The result is shown in FIG. 8B.

As illustrated, the result of the synthesis is that anions covalentlylinked to the surface 12 are disposed in position to interact withcations on the surface of the imaging protein, and hydrophobic groupsare positioned to interact by hydrophobic-hydrophobic attraction withthe hydrophobic patch on the protein. The hydrophobic amphipathicmolecules become bonded to the surface through secondary amine linkagesin precisely the same way as the lysine or arginine residues. Theamphipathic molecule may comprise, for example, a compound of theformula R-X, where R is a hydrophobic group such as a saturated orunsaturated hydrocarbon, halocarbon, either cyclic, branched, orstraight chained, or an aryl group or heterocyclic nucleus, and X is areactive group which will associate and can form covalent bonds with anappropriate activated surface. X may be, for example, an amine group asexemplified above, or a carboxylate group. When the activated surfacecomprises a field of amine groups, X may be an epoxy or aldehyde group.This procedure can be used with or without parallel formation of chargedgroups. It can also be used to produce imaged reverse phase matrixmaterials that selectively bind predetermined macromolecular species bymultipoint matching hydrophobic interaction.

FIGS. 9A-9C disclose the synthesis scheme for locating on the surface ofthe sorbent a metal coordinating compound, here iminodiacetic acid, at apoint designed to form a metal coordinating bond in the presence of ametal with an imidazole side chain. FIG. 9A illustrates an activatedsurface, here exemplified as an epoxy surface of the type in FIG. 3A,interfacing with a protein which, prior to the imaging step, has beenmixed with iminodiacetic acid and metal ion such as copper to form ametal coordination complex between the imidazole and IDA groups, shownin the center of FIG. 9A. Flanking the central histidine residue havingthe imidazole side group is a pair of lysine residues. Formation of theimaged surface occurs using a reaction scheme as illustrated in FIGS. 4Athrough 4E with the exception that a chelating agent such as EDTA isadded to the reaction mixture after hydrolysis of the protein to removethe coordinating metal, thereby leaving a iminodiacetic acid residueopposite in space from the location of the histidine residue on theprotein as illustrated in FIG. 9B. FIG. 9C schematically illustrates howthe image surface of 9B would interact with the imaging molecule. Asshown, in the presence of a metal such as copper (indicated in FIG. 9Cas M+) a metal coordinating bond forms between the imidazole residue andthe iminodiacetic acid residue at the same time as cations on thesurface of the protein and anions on the sorbent surface interact byelectrostatic attraction. This procedure also can be used above, to makeimaged metal coordinating matrix materials having multipoint metalcoordinating bond links, or together with the simultaneous production ofhydrophobic interactive functionalities.

The invention will be understood further from the following, nonlimitingexamples.

From the foregoing it will be appreciated that there are manyalternative strategies for producing a particular imaged surface, andthat, to optimize selectivity and affinity for a particular imagingmacromolecule, multiple syntheses may be desirable, with each synthesisfollowed by analysis to guide the next iteration, thereby to moreclosely attain the desired sorbent properties. An illustration of thisgeneral approach appears in the examples below.

It is contemplated in accordance with the invention that computer aideddesign will play an important role in facilitating development ofparticular imaged surfaces. An example of how software for modelingprotein and other macromolecular structure may be exploited to advantageis shown in FIG. 12. As illustrated, a given macromolecule, here"protein X" can simply be subjected to reactive imaging as disclosedherein to produce a "prototype" imaged surface. If the surface is to beused for low volume procedures, such as analysis, the prototype maysuffice. However, if structural data for protein X is known, itsthree-dimensional configuration and relevant surface properties may bediscerned using commercially available molecular modeling software in ageneral purpose computer. Thus, for example, depending on available dataon the macromolecule of interest, it may be possible to discover atleast the presence, approximate spacing, and relative positions of oneor more hydrophobic patches, histidine residues, or charged amino acidson particular surfaces on the macromolecule. This information may beused to aid the chemist in deciding which approach might be successfuland which would not, greatly decreasing the work involved. For example,whether metal chelating should be used, whether hydrophobic patchimaging alone may be successful, whether anti-chaotropic salts should beused in the imaging step, and if so, what face of the molecule likelywill be imaged, and what features are on that face, all can bedetermined by modeling. Thus, as in many engineering challenges,computer aided design techniques can give insights which streamline andshorten the design process.

In situations where a large volume of sorbent will be required, or whereit is desired to make a highly "monoclonal" imaged sorbent, inaccordance with the invention, one can find, analyze, and thensynthesize a peptide having a structure which mimics a surface of adesired macromolecule, and then can use the peptide in a reactiveimaging process. Alternative ways to implement this approach also aredisclosed in FIG. 12. Thus, peptides generated from partially hydrolyzedprotein X, some other protein source, or a synthetic peptide mixture,may be screened for binding to a prototype imaged surface, e.g., usingaffinity chromatography with differential elution using gradienteluents, to identify a peptide which binds to a prototype surface. Thispeptide then itself may be used to make a second surface, which in turnis tested for binding to protein X. If the new surface has the desiredbinding properties, large amounts of the imaging peptide may besynthesized and used to image production quantities of the imagedsorbent. If not, another peptide can be imaged, and the processrepeated.

Again CAD can benefit this process. Thus, analysis of a surface peptidesequence may reveal a surface mimicking peptide. It then can besequenced, synthesized, optionally screened on a prototype imagedsurface, and used in reactive imaging to produce a new surface. This newsurface can again be tested using protein X, as well as the identifiedpeptide or other peptides. Again, once a surface having the desiredproperties emerges, it can be duplicated in large volumes using thepeptide as the imaging macromolecule, and will bind selectively and withdesired affinity to protein X.

Example 1. Imaging lysozyme

POROS®--OH (PerSeptive Biosystems, Cambridge, Mass.) was preparedaccording to U.S. Pat. No. 5,030,352 using an epichlorohydrin-glycidolcopolymer that was subsequently crosslinked to produce a matrix rich insurface hydroxyl groups. This material was brominated with PBr₃ andsubsequently derivatized with sorbitol in the presence of strong base.The resulting surface, rich in diols, was oxidized with NaIO₄, thenimaged with lysozyme (30 mg/gram of beads) in the presence of 1.6M Na₂SO₄, 0.1M phosphate buffer (pH 9.0) for 20 hours in the presence ofNaCNBH₃. At the end of the reaction, excess aldehyde groups were reducedby NaBH₄. The bound protein was hydrolyzed with 4M KOH for 16 hours andsubsequently acylated with acetic anhydride for 2 hours. Excess estergroups were hydrolyzed for 2 hours with 0.5M KOH. The result was animaged sorbent of the type illustrated in FIG. 4E with anions on thesurface spaced in the mirror image of amine containing side groups onthe surface of lysozyme.

Fourier Transform Infrared Spectrospy (FTIR) was used to analyze theprogress of the reaction scheme. Thus, the generated spectra of lysozymealone, lysozyme immobilized onto the sorbitol activated surface,immobilized lysozyme with a spectrum of the base matrix subtracted out,the surface of the base matrix after hydrolysis, and surface matrixafter acylation, all were compared. The spectra of lysozyme alone andthe lysozyme on the base matrix were, as expected, very similar. Thespectrum after hydrolysis clearly illustrated the absence of lysozymewith loss of the characteristic maximum bands at 3300, 1650, and 1550cm⁻¹. Also, the spectrum of the support after acylation showed a pair ofbands at 1750 and 1200 cm⁻¹ corresponding to the presence of acetateesters. Lastly, the imaged support was titrated with 0.1M KOH in 5microliter increments. No measurable ionic capacity on the surface ofthe sorbent could be detected, indicating that anionic charge densitywas extremely low, as expected.

The sorbent then was packed in a 4.6×100 mm column, and cytochrome C andlysozyme were applied to the surface with a gradient of increasingsodium chloride from 0 to 1M at pH 6. A cytochrome C peak was elutedfrom the column at very low ionic strength, followed by a separate peakat only a slightly higher concentration indicating elution of lysozyme.

The conclusion from this experiment, showing rather weak lysozymebinding to the imaged surface, is that while a cation exchange surfacewas successfully manufactured as indicated by the behavior of the columnand by the FTIR spectra, based on the rather low salt concentration(approximately 0.3M) needed to elute the lysozyme in the gradient mode,the density of active groups on the surface of the starting material wasprobably too low, resulting in the creation of too few points of ionicinteraction between the surface of the lysozyme and the binding regionson the sorbent. Accordingly, in an attempt to improve affinity, theexperiment was repeated with a higher density of activated groups on thesurface of the starting material.

Example 2

Polystyrene divinylbenzene POROS® sorbent was again treated withepichlorohydrin-glycidol copolymer to produce a field of hydroxyl groupsabout the surface of the perfusive particulate material. This startingmaterial was then suspended in 750 milliliters of water, degassed byvacuum and nitrogen, and added to 25 ml acrolein and 12.5 g ceriumsulfate. The mixture was stirred for 8 hours at room temperature undernitrogen, then the beads were washed with water, sulfuric acid, water,and acetone, and dried in a vacuum oven at 60° C. This procedureresulted in the production of a aldehyde activated filamented surface ofthe type illustrated in FIG. 3B. The surface density of groups availableis much higher than the epoxy activated surface of FIG. 3A and Example 1as the aldehydes not only cover the surface of the POROS® support butalso extend from polymer filaments.

Thirty mg of lysozyme were dissolved in 2.5 ml of 0.1M phosphate buffer,pH 9.0. Next, 12.6 ml 2.0M sodium sulfate in 0.1M phosphate buffer, pH9, was mixed with the lysozyme, and the solution was added to 2 grams ofthe acrolein activated bead preparation. This mixture was shaken at roomtemperature for 3 hours, then 100 mg sodium borohydride added withshaking for another hour. The beads then were washed with water,suspended in 50 ml 4M KOH, and stirred with reflux for 16 hours. Thebeads were then washed with water and acetone and dried in a vacuum ovenof 60° C. Next, they were suspended in 25 ml pyridine followed byaddition of 25 ml acetic anhydride. The mixture was stirred under refluxfor 2 more hours, and the beads were then washed with water and acetoneand dried.

The progress of the synthesis was again traced with FTIR. The sameseries of infrared spectra were produced as discussed above with respectto Example 1. However, titration with 0.1 KOH revealed an ionic capacityof about 1 μM per ml.

As a control, the acrolein derivatized surface was exposed to thereaction scheme described above but in the absence of any imaging agent.Elution experiments on this type of material showed essentially nolysozyme retention on the control surface. Lysozyme could be eluted atless than 100 nM NaCl.

The chromatographic performance of the imaged surface was evaluated bypacking a 4.6×100 mm column with the material produced as disclosedabove. The plot of a gradient elution of lysozyme at pH 8 is shown inFIG. 10A. The column was loaded with a 100 μl injection of 1 mg/mllysozyme equilibrated with tris buffer at pH 8. A three minute gradientto 1M NaCl (27 mS conductivity) elutes the bound lysozyme at about 17mS.

The performance of this lysozyme imaged column can best be illustratedby comparison to a high capacity strong cation exchange column. FIG. 10Bshows the gradient separation of lysozyme and cytochrome C on acommercially available cation exchange column (POROS® HS/M). Lysozymeelutes at 0.55M NaCl (15 mS) while cytochrome C elutes at 0.42M NaCl,(11 mS, where 27 mS is approximately equal to 1M NaCl). In contrast,FIG. 10C shows the same test with the lysozyme imaged column. In thiscase, lysozyme elutes at 0.6M NaCl (16 mS) while cytochrome C elutes at0.15M NaCl (4 mS). The difference between these two surfaces is thatlysozyme is strongly bound to both but cytochrome C binds weakly to thelysozyme imaged column. The same separation profile on the imaged columnis illustrated when lysozyme is mixed with other proteins. FIG. 10Dshows that chymotrypsinogen binds weakly while lysozyme is boundtightly. FIG. 10E shows that lysozyme is bound tightly whileribonuclease is bound weakly.

Additional work suggests that these results are essentially duplicatedat pH 6.2. However, since the functional groups on the surface of theimaged column are weak anionic groups, i.e., carboxyl, one would expectthe column to lose its capacity with decreasing pH. As predicted, at pH4.5, the imaged column does not bind lysozyme well, eluting at thebeginning of the gradient, while cytochrome C is completely unretained.This contrasts with the strong cation exchanger used in FIG. 10B whichbinds both proteins well at pH 4.5.

If one seeks to use the lysozyme imaged column in an on/off affinitymode, a small amount of sodium chloride can be included in the sampleand wash buffers. In this case, the imaged column selectively bindslysozyme essentially exclusively from a mixture of lysozyme andcytochrome C provided the feed contains 100 mM sodium chloride. Elutionis then conducted by increasing sodium chloride content to 1.0M.

FIG. 11 shows a direct comparison of gradient elution between lysozymeand cytochrome-C conducted under precisely the same conditions, on alysozyme imaged column (top plot) and on a strong cation exchange (SCX)column (POROS® HS/M). While lysozyme binds strongly to both surfaces,cytochrome C, with a pI of 9, binds weakly to the imaged surface. Amodel of protein adsorption to ion exchange columns has been developedby Regnier et al. (see: The Role of Protein Structure in ChromatographicBehavior Science, Volume 238, page 319, Oct. 16, 1987). This modelcorrelates solute retention to the concentration of eluent by thefollowing equation: ##EQU1## where k' is a volumetric chromatographicretention factor, I is a constant related to the binding constant, D isthe concentration of salt used as eluent, and Z is a constant reflectiveof the number of interaction sites between the protein and the surface.

Isocratic experiments were performed to map the retention behavior oflysozyme and cytochrome C on strong cation exchange and on the imagedcolumn. Plots of log k' versus D for lysozyme and cytochrome C, followedby linear regression analysis allowed an estimate of the constants I andZ as shown in the table below.

                  TABLE I                                                         ______________________________________                                                Lysozyme         Cytochrome-C                                                 Imaged                                                                              SCX        Imaged  SCX                                          ______________________________________                                        I         0.25    0.03       0.006 0.003                                      Z         2.5     5.0        2.5   4.5                                        ______________________________________                                    

As shown, lysozyme interacts with a strong cation exchange surface (SCX)through five sites and with the imaged surface through only 2.5 sites.This indicates either binding through a different contact region on therespective surfaces (consistent with use of the anti-chaotropic salt inthe imaging process to salt out the lysozyme) or in low charge densitywith only 2 to 3 sites per molecule cross sectional area. The latterimplies that other solutes of similar size should interact with thissurface through at most 2.5 sites. This prediction is verified, at leastin the case of cytochrome C, which has a Z number of 2.5 on theantilysozyme imaged column.

Although lysozyme binds through only half as many sites on the imagedsurface as compared with the conventional strong cation exchange column,the binding constant with the imaged surface is about ten times higherthan on the strong cation exchanger. This provides strong evidence forcooperative binding with the imaged surface. The observation is furtherstrengthened by the large difference in ionic capacity between theimaged surface (1 μM/ml) and the strong cation exchange surface (50μM/ml). Finally, one can compare the relative binding strengths on thetwo surfaces. On the strong cation exchanger, lysozyme binds ten timesstronger than does cytochrome C, through an equivalent number of sites.Lysozyme binds 40 times as strongly to the imaged surface as doescytochrome C. Frontal loading experiments suggest the binding constantbetween lysozyme and the imaged surface, assuming a Langmuir isotherm todescribe the binding process, is about 2×10⁶ M⁻¹. Compared to antibodyantigen reactions, this is on the low end. However, given that only 2.5sites are involved, it represents a rather strong interaction.

Example 3

Bovine serum albumin (BSA) has a pI of 5.7. At pH 6 or 8, its surfacetherefore will be negative in charge. While BSA has surface amines thatcan be used in the reactive imaging process described in Example 2,electrostatic repulsion would be expected to interfere with theadsorption of BSA at such an imaged surface. With this premise, BSA wasimaged following the procedure of Example 2. FTIR spectra of thereaction scheme indicated that the imaging process has proceeded asexpected. The ionic capacity of the material was measured to be about1.7 μM/ml.

Chromatographic evaluation of this BSA imaged surface was performed inanalogous fashion to that described in Example 3. Elution profiles oflysozyme and cytochrome C from this surface showed that lysozyme isweakly retained, needing only about 0.2M NaCl to elute, while cytochromeC is unretained. As predicted, BSA binds to this BSA imaged surface onlyweakly. It is proposed that electrostatic repulsion is the reason forthis behavior. Thus, a cation exchange surface is formed which bindslysozyme through weak cation exchange, does not bind cytochrome C, andbinds its target poorly with the mechanistic explanation offered above.

Retention maps were generated to characterize the binding of lysozymeand cytochrome C to this BSA imaged surface in a way similar to that setforth above in Example 2. Binding of cytochrome-C to this surface wastoo weak to allow a reliable estimate of Z or I. The Z number forlysozyme was 4.2 while the I number was 0.004, and 0.003, respectively.The higher charge density, though not specific for lysozyme, does seemto increase the interaction sites to 4.2. The binding constant forlysozyme onto the BSA imaged surface is understandably low, comparableto that of cytochrome C on the lysozyme image surface.

Binding strength derives in part from the number of interaction sites.As discussed above, an overall binding strength number (I) is related tothe binding constant (K) between a solute and a surface ligand. Inexample 2, K was measured as 2×10⁶ M⁻¹ for lysozyme and the lysozymeimaged surface. Based on this measured K and the ratio of I values forvarious surfaces, one can determine K values for other related surfaces.Assuming that the overall binding constant is the product of individualinteraction constants, one can further calculate an average single sitebinding constant (K^(1/z)) as shown in the table below.

                  TABLE II                                                        ______________________________________                                        Estimated Average Single Site Binding Constant for                            Lysozyme on:                                                                  ______________________________________                                               SCX             115                                                           Lys-Imaged      330                                                           BSA-Imaged       12                                                    ______________________________________                                    

The foregoing analysis clearly shows the cooperative binding of lysozymeto the lysozyme imaged surface. While the BSA imaged and SCX surfacebehave similarly, the Lysozyme imaged surface binds 30 times strongerper interation site.

The invention may be embodied in other specific form. Accordingly, otherembodiments are within the following claims.

What is claimed is:
 1. A solid material defining a binding surfacecomprisinga coating adhered to a surface of said solid material anddefining a multiplicity of regions which selectively bind a preselectedorganic molecule having a plurality of ionizable groups spaced about amolecular surface thereof, each said region comprising a plurality ofcharged moieties bonded to said binding surface and disposed inspaced-apart relation within said region in a mirror image and chargeinverse of at least a subset of said ionizable groups whereby saidregions bind by spatially matched electrostatic attraction to themolecular surface of said preselected molecule preferentially to othermolecules.
 2. A composition of matter comprising:a solid materialdefining a binding surface comprising adhered coating defining amultiplicity of binding regions; and a multiplicity of organic moleculesbound by spatially matched multipoint electrostatic attractions to saidbinding regions, each said organic molecule defining a plurality ofionizable groups spaced about a molecular surface thereof, said bindingregions comprising a plurality of charged moieties bonded to saidbinding surface and disposed in spaced-apart relation within a saidregion in a mirror image and charge inverse of at least a subset of saidionizable groups.
 3. The invention of claim 1 or 2 wherein said solidmaterial comprises organic polymeric particulate material.
 4. Theinvention of claim 1 or 2 wherein said solid material comprises aperfusive matrix.
 5. The invention of claim 1 or 2 wherein said bindingsurface is substantially free of bound charged moieties in excess ofthose which bind to the ionizable groups of said organic molecule. 6.The invention of claim 1 or 2 wherein said charged moieties comprisenegatively charged moieties selected from the group consisting ofcarboxylate, sulfonate, phosphate and phosphonate moieties.
 7. Theinvention of claim 1 or 2 wherein said charged moieties comprisecarboxyl groups.
 8. The invention of claim 1 or 2 wherein said chargedmoieties comprise positively charged moieties selected from the groupconsisting of primary amines, secondary amines, tertiary amines, andquaternary ammonium.
 9. The invention of claim 1 or 2 wherein saidcharged moieties are bonded to said binding surface through flexiblemoieties and said spaced-apart charged moieties define aconformationally compliant charged surface.
 10. The invention of claim 9wherein said flexible moieties comprise oligomers varying in lengthwhereby individual said charged moieties are disposed varying distancesapart from said binding surface.
 11. The material of claim 1 whereinsaid preselected organic molecule defines a hydrophobic patch on saidmolecular surface, at least a subset of said regions furthercomprising:a hydrophobic moiety within said region at a locationdisposed to interface with said patch when said preselected molecule isbound to said region by multipoint electrostatic attraction.
 12. Thecomposition of claim 2 wherein said organic molecule further comprises ahydrophobic patch within said molecular surface and said binding regionscomprise a hydrophobic moiety which interfaces with said patch.
 13. Thematerial of claim 1 or 11 wherein said preselected organic moleculecomprises an imidazole moiety on said molecular surface and wherein atleast a subset of said regions further comprise a metal coordinatingmoiety bonded to said binding surface at a location in said regiondisposed to form, in the presence of coordinating metal ions, a metalcoordinating bond between said imidazole moiety and said coordinatingmoiety.
 14. The material of claim 13 wherein said coordinating moietycomprises an iminodiacetic acid moiety.
 15. The composition of claim 2or 12 wherein said organic molecule comprises an imidazole moiety onsaid molecular surface complexed through a metal ion to a metalcoordinating compound bonded to the surface of said regions.
 16. Theinvention of claim 1 or 2 wherein a subset of said binding regionscomprise spatial patterns of charged moieties in a mirror image andcharge inverse of at least a subset of ionizable groups spaced about asecond molecular surface of said organic molecule.
 17. The invention ofclaim 1 or 2 wherein said organic molecule comprises a peptide bondedamino acid polymer.
 18. The invention of claim 1 or 2 wherein saidmolecular surface and said regions have an interfacing area of at leastabout 50 square angstroms.
 19. The invention of claim 1 or 2 whereinsaid molecular surface and said regions have an interfacing area of atleast about 500 square angstroms.
 20. The invention of claim 1 or 2wherein said organic molecule comprises an analog of an intermediate inan organic reaction or a polypeptide analog of a surface region of amacromolecule.
 21. The invention of claim 1 or 2 wherein said bindingsurface is an oxygen rich polymeric hydrophilic surface.
 22. A solidmaterial defining a binding surface comprisinga coating adhered to asurface of said solid material and defining a multiplicity of regionswhich selectively bind a preselected organic molecule having animidazole moiety on a molecular surface thereof, each said regioncomprising a metal coordinating moiety bonded to said binding surfaceand disposed in said region in a mirror image position of said imidazolemoiety, whereby in the presence of coordinating metal ions, said regionbinds by multipoint spatially matched attractions including at least onemetal coordination bond between said imidazole moieties and saidcoordinating moieties, to the molecular surface of said preselectedmolecule preferentially to other compounds.
 23. The material of claim 22wherein said binding surface is substantially free of bound metalcoordinating moieties in excess of those which bind to the imidazolemoieties of said molecules.
 24. The material of claim 22 wherein saidmetal coordinating moiety is an iminodiacetic acid moiety.
 25. Thematerial of claim 22 wherein said organic molecule defines a hydrophobicpatch on said molecular surface, said region further comprising:ahydrophobic moiety within said region at a location disposed tointerface with said patch when said preselected polymer is bound to saidregion of said solid material.
 26. The material of claim 22 or 25wherein said preselected organic molecule comprises an ionizable groupon said molecular surface and said regions comprise a charged moietybound to said binding surface at a location opposite said ionizablegroup when said preselected molecule is bound to regions of said solidmaterial.
 27. The material of claim 22 or 25 wherein said regions bindan analog of an intermediate in an organic reaction or a polypeptideanalog of a surface region of a macromolecule.
 28. The material of claim22 wherein said organic molecule has plural imidazole moieties and eachsaid region comprises a plurality of said metal coordinating moieties.29. A solid material defining a binding surface comprisinga coatingadhered to the surface of said solid material and defining amultiplicity of regions which selectively bind a preselected organicmolecule having a hydrophobic patch on a molecular surface thereof, eachsaid region comprising at least one hydrophobic moiety, surrounded byhydrophilic surface, bonded to said binding surface, and disposed withinsaid region in a mirror image position to said hydrophobic patch,whereby said regions bind by spatially matched attractions including atleast one hydrophobic-hydrophobic interaction, to the molecular surfaceof said preselected compound preferentially to other compounds.
 30. Thematerial of claim 29 wherein said preselected organic molecule comprisesan imidazole moiety on said molecular surface and wherein at least asubset of said regions further comprise a metal coordinating moietybonded to said binding surface at a location in said region disposed toform, in the presence of coordinating ions and when said preselectedmolecule is bound to said regions, a metal coordinating bond betweensaid imidazole moiety and said coordinating moiety.
 31. The material ofclaim 29 or 30 wherein said preselected organic molecule comprises anionizable group on said molecular surface and said regions comprise acharged moiety bound to said binding surface at a location opposite saidionizable group when said preselected molecule is bound to regions ofsaid solid material.
 32. The material of claim 29 wherein said organicmolecule has plural hydrophobic patches spaced about the molecularsurface thereof and at least a subset of said regions comprise pluralsaid hydrophobic moieties in mirror image position to bind with saidpatches.
 33. The material of claim 29 wherein said region binds ananalog of an intermediate in an organic reaction or a polypeptide analogof a surface region of a macromolecule.
 34. A solid material defining abinding surface comprising a coating adhered to the surface of saidsolid material and defining a multiplicity of regions which selectivelybind a preselected macromolecule having one or more of at least twosurface features, spaced about a molecular surface thereof, selectedfrom the group consisting of ionizable moieties, hydrophobic patches,and imidazole moieties,each said region comprising, in a hydrophilicfield, a plurality of moieties selected from the group consisting ofcharged moieties, hydrophobic moieties, metal coordinating moieties, andcombinations thereof, bonded to said binding surface and disposed inspaced-apart relation within said region in a mirror image of at least asubset of said at least two surface features, whereby said regions bind,by at least two of a) spatially matching electrostatic attractionbetween said ionizable moieties and said charged moieties, b) metalcoordination in the presence of coordinating metal ions between saidimidazole moieties and said metal coordination moieties, and c)hydrophobic-hydrophobic interaction between said hydrophobic patches andsaid hydrophobic moieties, to the molecular surface of said preselectedmacromolecule preferentially to other polymers.